Hydrogels are three-dimensional networks that include a polymeric continuous phase that can retain a significant volume of water as the dispersed phase and as such resemble the extracellular matrix (ECM) of soft tissues. Hydrogels have been found useful as matrix materials in tissue engineering due to their high diffusivity of, e.g., nutrients and biomolecules. As successful tissue generation will include the formation of new tissue, useful tissue engineering hydrogels must be capable of degradation in order to provide necessary free volume for the newly generated tissue. Synthetic hydrogels that allow for a high water content such as those based on polyvinyl alcohol (PVA), polyhydroxyethyl methacrylate (PHEMA), polyethylene glycol (PEG) and polyvinylpyrrolidone (PVP) have been examined for use in tissue engineering applications. Although synthetic hydrogels can provide matrices with tunable properties, they have little resemblance either structurally or chemically to the natural ECM. Moreover, viability and fate of encapsulated cells in such hydrogels have been limited by the toxic effect of gelation and degradation reactions of the polymer matrix.
Consequently, natural hydrogels derived from components of the ECM that crosslink and degrade enzymatically with no toxic side effects have been preferred for use in clinical applications such as tissue generation. For instance, injectable hydrogel-forming compositions based on natural polymers that are capable of in-situ gelation are highly attractive for use in minimally-invasive arthroscopic procedures, such as in delivery of regenerative cells to irregularly-shaped reconstruction sites. To date, injectable hydrogel forming compositions based on collagen, gelatin and composites thereof that include other natural biopolymers have been used as injectable gels for cell encapsulation and delivery in tissue regeneration. Gelatin, which is produced by partial hydrolysis of collagen, is a mixture of proteins with a wide range of molecular weights. Beneficially, hydrogels based on natural ECM polymers can include amino acid sequences involved in cell-matrix interaction for adhesion, growth, differentiation, and maturation of the encapsulated cells. Unfortunately however, collagen-based hydrogels suffer from several issues that prevent their wider successful use such as batch-to-batch variability in composition, limited thermal and mechanical stability, and relatively fast and uncontrollable enzymatic degradation in vivo.
Keratin is an abundant natural protein found in poultry feather, animal hair and horn, and human hair. Due to its high strength and biocompatibility, keratin-based membranes, sponges, and fiber meshes have been developed as scaffolds for tissue engineering applications. Keratin has a relatively high fraction of cysteine residues (generally about 7 mol % to about 20 mol % of the total amino acid content) compared to other proteins, and partial alkylation of sulfhydryl groups of the cysteine residues combined with freeze drying and crosslinking have been used to produce porous keratin hydrogel scaffolds with tunable pore sizes and stiffness for e.g., cell seeding. Unfortunately, keratin-based hydrogel formation techniques do not form a material capable of injection and in situ crosslinking and are thus not compatible with many clinical procedures, for instance for injection in a precursor form in conjunction with a cell suspension followed by in situ crosslinking.
There is a need for natural protein-based hydrogels that include features of the natural ECM with predictable amino acid composition that can be formed with predetermined degradation control and porosity characteristics. Moreover, an injectable precursor composition capable of in situ crosslinking following injection to form the hydrogels at a target site would be of great benefit.